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In this work, two gun barrels were 3D‐printed using a metal alloy material from the same 3D digital model created insilico. After assembling the barrels in a reference Colt 45 Auto semi‐automatic pistol, a total of 100 rounds of ammunition were successfully test‐fired through each barrel. Heavy and gross striations were observed on all fired bullets. The striations on bullets discharged from the corresponding 3D‐printed gun barrel were found to be identifiable. Moreover, bullets fired from one 3D‐printed gun barrel were easily excluded from those fired from the other 3D‐printed gun barrel by visual examination under a comparison microscope. The resulting unconventional striations that were observed were apparent to an experienced firearm and toolmark examiner. These features could provide valuable investigative leads related to the 3D printing process. Since 3D printing has become an option for firearm manufacturing, the forensic science community should establish a knowledge base associated with the toolmark features generated by the 3D‐printed products. [ABSTRACT FROM AUTHOR]